The generation of the Aa-peptide requires the cleavage of its precursor A4CT (C99) by the so-far unidentified (-secretase. Since the position of (-secretase cleavage is crucial for the pathogenesis of Alzheimer's disease, we individually replaced all membrane domain residues of A4CT outside the Aa-domain by phenylalanine, stably transfected the constructs in COS7-cells and determined the effect of these mutations on the cleavage specificity of (-secretase (Aa42/Aa40-ratio). Compared to A4CT-wildtype, mutations at Val44, Ile47 and Val50 led to decreased Aa42/Aa40-ratios whereas mutations at Thr43, Ile45, Val46, Leu49 and Met51 led to increased Aa42/Aa40-ratios. The strongest effect was observed for Ile45Phe (34-fold increase) making this construct highly valuable for the generation of animal models for Alzheimer's disease. Unlike the other mutations, A4CT-V44F was mainly processed to Aa38 as determined by mass spectrometry. Our data provide for the first time a detai led possib le model for the active site of (-secretase: (-secretase interacts with the transmembrane domain of A4CT by binding to one side of the ?-helix. Mutations in the transmembrane domain of A4CT interfere with the interaction of (-secretase and A4CT. As a result, the cleavage specificity of